Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

The effect of chlorpromazine on the temperature and osmotic sensitivity of erythrocytes]

The effect of chlorpromazine on the development of cold shock in erythrocytes exposed to sodium chloride was shown to depend on the tonicity of the medium in which the cells were cooled from 37 degrees C down to 0 degrees C as well as on the amphipate concentration. After cooling of erythrocytes in a NaCl (0.75-1.5 M)-containing medium with chlorpromazine (7 x 10(-5) M, 2.1 x 10(-4) M and 3.5 x 10(-4) M) the hypertonic cold shock was inhibited, the protective effect of the amphipate being less pronounced at its increasing concentrations. After cooling of cells under conditions of moderate hypertonicity (0.3-0.6 M NaCl) no modifying effect of chlorpromazine on the sensitivity of erythrocytes to the temperature decrease from 37 degrees C down to 0 degrees C was manifested. However, under iso- and hypertonic conditions chlorpromazine used at 2.1 x 10(-4) M and 3.5 x 10(-4) M stimulated the cold shock development in erythrocytes. A sharp increase in the medium tonicity (up to 1.8-3.0 M and higher) the cells underwent isothermal hemolysis which was more expressed at 0 degrees C than at 37 degrees C. These data suggest that chlorpromazine significantly activates the hemolytic process at low temperatures.     

Membrane ordering effects of the anticancer agent VM-26

The effect of the anticancer agent VM-26 on acyl chain order of cellular and model membranes was examined by electron spin resonance techniques. The order parameter for the paramagnetic probe 5-doxyl stearate was increased when VM-26 was incorporated into the bilayer of fluid-phase dimyristoylphosphatidylcholine (DMPC) or gel-phase dipalmitoylphosphatidylcholine (DPPC) liposomes at concentrations up to 4.8 mol%. The ordering effect of VM-26 in DMPC was greater than that of cholesterol on an equimolar basis. The less cytotoxic congener of VM-26, VP-16, was only one-third as active as VM-26 in its ordering effects on DMPC. Higher order parameters for 5-doxyl stearate were also noted in asolectin liposomes, Ehrlich ascites tumor cells, and CCRF-CEM cells treated with VM-26. We conclude that VM-26 has significant membrane associated activity in addition to its previously recognized nuclear effects.     

Measurement of the water permeability of single human granulocytes on a microscopic stopped-flow mixing system

The water permeability (Lp) of human granulocytes was measured for individual isolated cells with a novel, microscopic stopped-flow mixing system. Changes in volume were monitored as a cell was introduced suddenly into an osmotically active solution. Permeability values were determined as a function of solution osmolality from the volume versus time curves for mixing into both hypotonic and hypertonic solutions within the range of 145 to 833 mOsm. The calculated reference permeability at 25 degrees C was 1.15 micrometers/atm.min with an osmotic coefficient of 0.46 Osm/kg.     

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.     

Thermal shock hemolysis in human red cells. I. The effects of temperature, time, and osmotic stress

Thermal shock is a form of hemolysis which occurs in human red cells exposed to greater than a critical level of osmotic stress of 1.4 Osm and subsequently cooled from above about 12 degrees C to below that temperature. Higher concentrations and higher cooling rates each increase the amount of hemolysis, within limits. Incubation for varying periods in hypertonic solutions and varying temperatures of incubation affect the amount of thermal shock. The effect of cooling rate on thermal shock is independent of the period of exposure to hypertonic solutions. Thermal shock is not the cause of freezing injury in human red cells, at least above -10 degrees C.     

Lipid loss and haemolysis by thermal shock: Lack of correlation

Haemolysis by thermal shock was unaffected by altering the solute cation but was dependent on solute anions. This suggests that cellular shrinkage is not the critical factor for the induction of thermal shock. Both glycerol and DMSO reduce thermal shock damage in hypertonic sodium chloride. The effect of time of exposure to hypertonic solutions observed at 37 °C was not affected by the metabolic inhibitors ouabain, flouride and PCMBS. The only additive to have any significant effect was phloretin.No evidence was obtained for the loss of membrane lipids or proteins from intact erythrocytes. Under each of the hypertonic conditions studied there appeared to be a correlation between the loss of membrane lipid and cellular lysis at constant temperature before cooling. There does not appear to be any correlation between the ratio of phospholipid to cholesterol in the hypertonic solution (a possible function of the membrane phospholipid:cholesterol ratio) and lysis upon a subsequent reduction in temperature.The protective effect of egg lecithin against thermal-shock damage in hypertonic solutions was confirmed; phosphatidyl serine was also found to be effective in reducing thermal shock. Phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin had no effect.     

Thermal shock and dilution shock as the causes of freezing injury

We suggest that during slow freezing, cellular membranes are altered by the hypertonic solutions produced. This alteration in itself does not cause membrane leakage of normally impermeant solutes but it renders the cells susceptible to solute leakage on the application of a stress, which is provided during freezing by the reduction in temperature (thermal shock) and during thawing by dilution (dilution shock).During slow freezing the effects of cooling rate changes are due to the different times available for the hypertonic solutions to affect the membrane. At a given cooling rate cryoprotective agents reduce the effect on the cells at each temperature during freezing perhaps by reducing the ionic strength. The thermal shock stress during cooling and the dilution shock during thawing thus damages the cells less. With rapid freezing, there is insufficient time for these effects to take place during cooling, which allows the cells to reach low temperatures without thermal shock damage. However, the presence of extracellular ice and the formation of intracellular ice provide hypertonic conditions that render the cells liable to dilution shock on thawing. The slower the rate of thawing of rapidly cooled cells the greater will be the damage from this dilution shock.     

Effect of osmotic stress on the ultrastructure and viability of the yeast Saccharomyces cerevisiae

Exposure of the yeast Saccharomyces cerevisiae to hypertonic solutions of non-permeating compounds resulted in cell shrinkage, without plasmolysis. The relationship between cell volume and osmolality was non-linear; between 1 and 4 osM there was a plateau in cell volume, with apparently a resistance to further shrinkage; beyond 4 osM cell volume was reduced further. The loss of viability of S. cerevisiae after hypertonic stress was directly related to the reduction in cell volume in the shrunken state. The plasma membrane is often considered to be the primary site of osmotic injury, but on resuspension from a hypertonic stress, which would have resulted in a major loss of viability, all cells were osmotically responsive. The effects of osmotic stress on mitochondrial activity and structure were investigated using the fluorescent probe rhodamine 123. The patterns of rhodamine staining were altered only after extreme stress and are assumed to be a pathological feature rather than a primary cause of injury. Changes in the ultrastructure of the cell envelope were examined by freeze-fracture and scanning electron microscopy. In shrunken cells the wall increased in thickness, the outer surface remained unaltered, whilst the cytoplasmic side buckled with irregular projections into the cytoplasm. On return to isotonic solutions these structural alterations were reversible, suggesting a considerable degree of plasticity of the wall. However, the rate of enzyme digestion of the wall may have been modified, indicating that changes in wall structure persist.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Role of cytoplasmic proteins in cold shock injury of Amoeba

Strains of Amoeba have been used to study the mechanisms of cellular injury induced by rapid cooling (cold shock). Cell viability was found to depend on the time and temperature of cold exposure, on the rate of cooling and on the morphology of the cells prior to chilling. All strains underwent a granuloplasmic contraction following undercooling to ?10 °C, although its extent varied; strains most damaged by cold shock exhibited the most violent cytoplasmic contractions. Cryomicroscopy demonstrated that the cellular contraction occurred upon rewarming, not during cooling. Cells damaged by cold shock were osmotically responsive, demonstrating that irreversible damage to the plasmalemma does not account for the phenomenon.Several compounds protected Amoeba against cold shock injury, glycerol and glucose being the most effective. With glycerol an optimum rate of cooling was observed upon cooling to ?10 °C, at both faster and slower cooling rates damage increased.The state of cellular actin in control cells and following cold shock was monitored by the DNase 1 inhibition assay and by electron microscopy. A comparatively “cold shock resistant” strain of A. proteus was found to contain less total actin per unit cellular protein than the more “sensitive” Amoeba sp. strain Bor. In the Bor strain a cold-induced aggregation of cytoplasmic filaments was evident in electron micrographs, presumably a crosslinking of preexisting F-actin.     

An L-Form of Staphylococcus aureus Adapted to a Brain Heart Infusion Medium without Osmotic Stabilizers

An L-form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L-form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, d-cycloserine, and vancomycin of the L-form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L-form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L-form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall-synthesis, than the original L-form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L-form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 μ/mg).     

Multifaceted freezing injury in human polymorphonuclear cells at high subfreezing temperatures

Extracellular freezing injury at high subzero temperatures in human polymorphonuclear cells (PMNs) was studied with a cryomicroscope, electron microscope, and functional assays (phagocytosis, microbicidal activity, and chemotaxis). There are at least four major factors in freezing injury: osmotic stress, chilling, cold shock, and dilution shock. Extracellularly frozen PMNs lose functions when cooled to -2 degrees C without a cryoprotectant. Cells lose volume on freezing to the same degree as in hypertonic exposure. PMNs have a minimum volume to which they can shrink without injury. Greater dehydration produces irreversible injury to cellular functions, and cells eventually collapse under high osmotic stress. Chilling sensitivity is seen in slowly chilled, supercooled PMNs below -5 degrees C; at -7 degrees C, functions are lost in 1 h. This injury can be prevented by the addition of Me2SO but not glycerol. Me2SO does not, however, prevent cold shock (injury due to rapid cooling), which is seen during cooling at 10 degrees C/min to -14 degrees C, but not during slow cooling at 0.5 degrees C/min. One of the problems of using glycerol as a cryoprotectant stems from the high sensitivity of PMNs to dilution shock during the dilution or removal of glycerol.     

Effects of cooling rate on thermal shock hemolysis

The increase in thermal shock hemolysis in hypertonic sodium chloride with increasing cooling rate was confirmed. Thermal shock damage was also induced by hypertonic solutions of sucrose but it decreased with increasing cooling rate. The effect of cooling rate on thermal shock hemolysis appears to be due to the time that the cells are in the hypertonic solutions. The extent of the stress of the temperature reduction was independent of the cooling rate. In hypertonic sodium chloride susceptibility to thermal shock damage increased with increasing time of exposure at +25 °C (0–5 min) before decreasing with time (5–50 min). In contrast, with hypertonic sucrose, thermal shock damage increased gradually with time of exposure. The protective effects of sucrose on thermal shock hemolysis at a given osmolality can be explained by the different solution properties (e.g., ionic strength) of hypertonic sodium chloride and sucrose. These results suggest that the role of thermal shock damage during slow freezing should be reexamined.     

Cold and hyperosmolar fluids in canine trachea: vascular and smooth muscle tone and albumin flux

We have studied the effects of liquids of various osmolalities and temperatures on the tracheal vasculature, smooth muscle tone, and transepithelial albumin flux. In 10 anesthetized dogs a 10- to 13-cm length of cervical trachea was cannulated to allow instillation of fluids into its lumen. The cranial tracheal arteries were perfused at constant flow, with monitoring of the perfusion pressures (Ptr) and the external tracheal diameter (Dtr). Control fluid was Krebs-Henseleit solution (KH) with NaCl added to result in a 325-mosM solution (isotonic). Hypertonic solutions were KH with NaCl (warm hypertonic) or glucose (hypertonic glucose) added to result in a 800-mosM solution. All solutions were at 38 degrees C, with isotonic and the hypertonic NaCl solutions also given at 18 degrees C (cold isotonic and cold hypertonic). Fluorescent labeled albumin was given intravenously, and the change in fluorescence in the fluid was measured during each 15-min period. Changing from warm isotonic to cold isotonic decreased Dtr and Ptr. Changing from warm isotonic to warm hypertonic or hypertonic glucose decreased Ptr with no change in Dtr. The cold hypertonic responses were not different from cold isotonic responses. Warm hypertonic solution increased albumin flux into the tracheal lumen over a 15-min period to three times that of the control period, persisting for 15 min after replacement with warm isotonic solution. Cooling induces a vasodilation and smooth muscle contraction of the trachea, whereas hypertonic solutions result in vasodilation and, if osmolality is increased with NaCl, an increase in albumin flux into the tracheal lumen.     

Rotational relaxation of 1,6-diphenylhexatriene in membrane lipids of cells acclimated to high and low growth temperatures.

Measurement of the time-resolved fluorescence depolarization of 1,6-diphenylhexatriene (DPH) in artificial bilayers of microsomal membrane lipids from Tetrahymena gives detailed information concerning the molecular motion of this probe and fluid properties of the membrane lipids which are obscured with steady-state methods. The rotational motion of DPH in these lipids from cells acclimated to 15 and 39.5 degrees C growth temperatures was anisotropic, which agrees with recent time-resolved studies of this probe in synthetic phospholipid systems. Evaluation of DPH polarization data obtained from these lipid fractions at their respective growth temperatures showed differences in physical properties which suggest that "viscosity", per se, of the microsomal lipids is not a strictly regulated as it is in prokaryotic systems. Rotational relaxation of DPH in 39.5 degrees C microsomal lipids measured at 15 degrees C is more complex than that of either lipid fraction measured at its actual growth temperature, suggesting that the probe has partitioned into two dissimilar environments within the bilayer. Similar effects are observed in the microsomes of 39.5 degrees C cells by freeze-fracture electron microscopy following rapid cooling to 15 degrees C. Under these conditions, two distinct regions are observed on the fracture faces, suggesting a correlation between lipid phase changes and alterations in membrane structure.     

Some combined effects of hypertonic solutions and changes in temperature on posthypertonic hemolysis of human red blood cells

The combined effects of hypertonic solutions and temperature changes on the posthypertonic hemolysis of human red blood cells have been investigated. Cells were exposed to hypertonic solutions of sodium chloride and also to hypertonic solutions of the extracellular cryoprotective additive sucrose, such as would occur during the freezing of cells in an isotonic salt solution to which 15% $\text{w}\text{v}$ sucrose had been added. In both cases the extent of posthypertonic hemolysis was increased by temperature reduction per se when the osmolality of the extracellular solution exceeded about 1400 mOsm/kg water. The posthypertonic hemolysis of cells exposed to a hypertonic solution at 0 °C was reduced with the temperature of the resuspension solution up to 35 °C.     

Factors influencing survival of mammalian cells exposed to hypothermia. II. Effects of various hypertonic media

Survival of Chinese hamster lung (V79) cells, exposed as a function of time to hypothermia in tissue culture, in isosmotic and various hypertonic media was measured using a colony assay. The mechanism of hypothermic cell killing is different above and below 7 degrees C in this cell line. Addition of NaCl or mannitol to increase the tonicity to 400 mOsm greatly decreased the survival at 10 degrees C while addition of KCl had no significant effect. When these experiments were repeated at 5 degrees C, addition of either NaCl, KCl, or mannitol was detrimental to long-term cell survival. Furthermore, addition of mannitol to the medium did not improve survival when cells were stored at 7 degrees C. Addition of KCl at 5 or 10 degrees C or NaCl at 5 degrees C only affected the cells' ability to accumulate sublethal damage, while addition of mannitol at 5 or 10 degrees C affected both of the above and the cold sensitivity of the cells. Addition of NaCl at 10 degrees C only affected the latter. These experiments suggest that prevention of cell swelling by these conditions, while possibly necessary during clinical hypothermic organ storage, is detrimental to single cell survival at these temperatures.